Colony-stimulating factors are glycoproteins that stimulate the growth of hematopoietic progenitors and enhance the functional activity of mature effector cells. Human GM-CSF is a 22-kDa glycoprotein that stimulates the growth of myeloid and erythroid progenitors in vitro and increases the responsiveness of neutrophils, monocytes, and eosinophils to physiologic stimuli.
Wong et al., Science Vol. 228, pp. 810-815 (1985) and Kaushansky et al., Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 3101-3105 (1986) have described the production of recombinant GM-CSF in mammalian cells. Burgess et al., Blood, Vol. 69, pp. 43-51 (1987) describes the purification of GM-CSF produced in Escherichia coli.
The extensive application of recombinant methodolgies to the large scale preparation of eucaryotic proteins has substantially enhanced the prospects for obtaining desired molecules in quantity and in some instances even simplified purification procedures needed to obtain biologically active materials. Illustratively, where the desired recombinant proteins need not be glycosylated to possess biological activity, large quantities of protein can often be produced in E. coli recombinant hosts in the form of insoluble "inclusion bodies" which contain few proteinaceous contaminants, proteases, or the like. Host cell lysates frequently include proteinaceous constituents of sufficiently similar molecular weight, charge, polarity and solubility characteristics (vis-a-vis the recombinant protein) to make ready separation difficult. Further proteolytic enzymes endogenous to the host provide a relatively chronic source of biological activity loss for the desired protein.
In accordance with the procedure described by Burgess et al., bacterially produced GM-CSF was lysed from the microorganism, suspended in guanidinium hydrochloride and mercaptoethanol and chromatographed over G-100 Sephadex. The fractions were collected and the denaturant and reductant removed by dialysis against neutral Tris buffer. The final purification step was gradient elution from a reverse-phase support at pH 2.1.